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Am J Physiol Heart Circ Physiol
280: H2726–H2731, 2001.
Cardiomyocyte apoptosis and ventricular remodeling
after myocardial infarction in rats
EEVA PALOJOKI,1,2 ANTTI SARASTE,3,4 ANDERS ERIKSSON,1,2 KARI PULKKI,5 MARKKU
KALLAJOKI,6 LIISA-MARIA VOIPIO-PULKKI,2 AND ILKKA TIKKANEN1,2
1
Minerva Institute for Medical Research, Departments of 2Medicine and 5Clinical Chemistry,
Helsinki University Central Hospital, Helsinki FIN-00029; and Departments of 3Anatomy,
4
Medicine, and 6Pathology, University of Turku, Turku FIN-20520, Finland
Received 23 November 1999; accepted in final form 23 January 2001
myocyte apoptosis has recently been shown to occur in
ischemic and reperfused myocardium both in animal
models (1, 5, 8, 13) and in humans (12, 24). Moreover,
apoptosis has been found in viable myocardial areas
after MI (3, 21, 24) and in experimental (26) and
human ischemic heart failure (17, 19, 25).
To clarify the role of apoptosis in chronic post-MI
remodeling, we studied the time course and anatomic
distribution of cardiomyocyte apoptosis in rat MI. We
assessed the occurrence of cardiomyocyte apoptosis
from 24 h to 12 wk after infarction in the infarcted,
border zone and remote noninfarcted myocardial regions and compared the proportions of apoptotic myocytes with echocardiographic measures of the remodeling process.
MATERIALS AND METHODS
(LV) remodeling after myocardial infarction (MI) involves expansion of the infarcted area,
ventricular dilatation, and thinning of the ventricular
wall (20, 22, 28). Cellular mechanisms of this process
include myocyte hypertrophy, elongation, and topographic rearrangements, such as side-to-side slippage
(20).
Apoptosis is a distinct type of cell death characterized by a series of typical morphological events, such as
shrinkage of the cell, fragmentation into membranebound apoptotic bodies, and rapid phagocytosis into
neighboring cells without induction of inflammatory
response (15). The biochemical hallmark of apoptosis is
internucleosomal DNA fragmentation (30). Cardio-
Experimental MI and tissue sampling. MI was produced by
ligation of the left anterior descending coronary artery.
Briefly, adult male Wistar rats weighing 350–500 g were
anesthetized subcutaneously by using 0.5 mg/kg of medetomidine (Domitor, Orion; Turku, Finland) and with 70–80
mg/kg ip ketamine (Ketalar, Parke-Davis; Barcelona, Spain).
The rats were connected to a respirator through a tracheotomy, and the heart was rapidly exteriorized through a left
thoracotomy and pericardial incision. The coronary artery
was ligated about 3 mm from its origin, the heart was
returned to its normal position, and the thorax was closed.
Throughout the operation, the body was maintained at a
stable temperature with the use of a thermal plate. The
anesthesia was partially antagonized with atipamezole hydrochloride (0.75 mg/kg sc, Antisedan; Orion), and the rats
were disconnected from the respirator. The rats were hydrated postoperatively with 10 ml sc of physiological saline
and given 0.02 mg/kg sc of buprenorphine hydrocholoride
(Temgesic; Reckitt and Colman; Hull, UK) twice for analgesia. The control rats underwent the same procedure except
for the ligation of the coronary artery (sham operation).
After 24 h, 1 wk, 4 wk, and 12 wk after coronary ligation,
the rats were euthanized with CO2 (n ⫽ 6–14 rats in each
group). The heart was excised and cut into 2-mm transverse
slices below the point where the coronary artery was ligated.
The myocardial samples were fixed in 4% neutral-buffered
Address for reprint requests and other correspondence: L.-M. Voipio-Pulkki, Emergency Care, Dept. of Medicine, Helsinki Univ. Central Hospital, PO Box 340, FIN-00029 HUS, Finland (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
ischemia
LEFT VENTRICULAR
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0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.ajpheart.org
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Palojoki, Eeva, Antti Saraste, Anders Eriksson, Kari
Pulkki, Markku Kallajoki, Liisa-Maria Voipio-Pulkki,
and Ilkka Tikkanen. Cardiomyocyte apoptosis and ventricular remodeling after myocardial infarction in rats. Am J
Physiol Heart Circ Physiol 280: H2726–H2731, 2001.—We
investigated the role of cardiomyocyte apoptosis in the remodeling of the left ventricle from 24 h to 12 wk after
myocardial infarction in the rat. Infarct size planimetry,
quantification of cardiomyocyte apoptosis, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling
(TUNEL) methodology, and echocardiography (left ventricular diastolic diameter and ejection fraction) were performed.
Sham-operated animals showed low rates of cardiomyocyte
apoptosis (0.03%) and no change in diastolic diameter or
ejection fraction during the study. Twenty-four hours after
infarction, TUNEL positivity was high in the infarct areas
(1.4%) and border zones (4.9%). It declined to 0.34% (P ⬍ 0.01
vs. sham) at 4 wk and 0.10% at 12 wk in the border zones. In
the remote myocardium, cardiomyocyte apoptosis increased
to 0.07% (P ⫽ 0.03 vs. sham) on day 1 and remained on the
same level up to 4 wk. The increase in diastolic diameter 1–4
wk after infarction correlated (r ⫽ 0.60, P ⬍ 0.01) with
cardiomyocyte apoptosis in the noninfarcted myocardium,
which quantitatively contributed most (⬎50%) to the apoptotic cell loss by 4 wk.
APOPTOSIS AND VENTRICULAR REMODELING
counted in the infarcted tissue, in the tissue bordering infarction (1-day infarctions), in the border zones of infarct
scars (1- to 12-wk infarctions), and in the remote noninfarcted myocardium. The myocardium extending 0.5–1.0 mm
from the infarcted tissue or infarct scar was considered to
represent the border zone myocardium. To avoid contamination of the remote myocardium with border zones, a myocardial area extending ⬃1–2 mm from the border zone area was
not included in the statistical analysis. The rest of the LV
was considered to represent the remote myocardium. To
assess the anatomical distribution of apoptotic cells quantitatively, we determined the relative proportions of the infarcted, border zone and remote myocardial regions of the
total area of the LV in the analyzed section.
Statistical analysis. Quantitative results were calculated
as means ⫾ SD. The differences in the amounts of apoptotic
cardiomyocytes between groups were compared using oneway ANOVA and Bonferroni’s method (SPSS Software; Chicago, IL). Echocardiography measurements between baseline
and end point were compared in each group with the use of
Student’s t-test for paired data. Pearson’s correlation coefficients were calculated to compare the amounts of TUNELpositive cells and echocardiography measurements.
RESULTS
Myocardial infarction. Histologically, there was either an area of necrotic myocardium (day 1 after operation) or a collagenous infarct scar (1–12 wk after the
operation) in 80% of the rats that underwent coronary
artery ligation. Planimetrically, there were small,
moderate, and large infarctions at a rate of 41, 28, and
31%, respectively. The sizes were equally distributed
at all time points.
Cardiomyocyte apoptosis. Apoptotic cells were rarely
found in the sham-operated animals with the use of the
TUNEL assay. In contrast, after coronary artery ligation, the apoptotic cells were much more numerous.
TUNEL-positive inflammatory cells and interstitial
cells were frequently observed in the infarcted areas
24 h after coronary ligation and among the scar tissue
1–12 wk after infarction. In contrast, very few scattered TUNEL-positive inflammatory or interstitial
cells were found in the border zones of infarct scars and
the remote myocardium. Also, TUNEL-positive cardiomyocytes were numerous 24 h after coronary ligation
in the infarcted tissue (Fig. 1A). At later time points,
we observed scattered TUNEL-positive cardiomyocytes
in the border zones adjacent to infarct scars and in the
remote myocardium (Fig. 1, B and C), whereas they
were absent among the scar tissue. The TUNEL-positive cardiomyocytes contained condensed nuclei, which
is a typical feature of cells undergoing apoptosis (Fig. 1,
A and B).
Quantitatively, the percentage of apoptotic cardiomyocytes in the myocardium of sham-operated rats
was on average only 0.03 ⫾ 0.02% and remained stable
during the study (Fig. 2). Compared with sham-operated rats, significantly higher percentages were found
after ligation of the coronary artery. On day 1 after
ligation, an average of 4.93 ⫾ 2.51% of TUNEL-positive
cardiomyocytes were found in the border zones of infarcted myocardium. Moreover, clusters of positive
cells were found among the necrotic myocytes in the
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formalin for 24 h, embedded in paraffin, and cut in 4-␮mthick sections for histology, planimetry, and assessment of
apoptosis.
Echocardiography. All of the experimental animals underwent echocardiography under anesthesia just before the operation (baseline) and 24 h, 1 wk, and 4 wk after coronary
ligation or sham operation. The animals were sedated with
medetomidine and placed on a warm thermal plate. The
stability of the body temperature was monitored with the use
of a rectal probe. The echocardiographic measurements were
performed with the use of a 12-MHz ultraband sector probe
(SONOS model 5500, Hewlett-Packard; Andover, MA). LV
systolic diameter and LV diastolic diameter (LVDD), respectively, were measured in the short-axis M-mode right
parasternal projection in a plane below the mitral valves and
perpendicular to the LV (27). An average of five measurements were used to measure LVDD and to calculate ejection
fraction (EF). The coefficients of variation for repeated measurements of LV systolic diameter and LVDD were 0.027 and
0.046, respectively.
Histology and planimetry of infarct size. The presence of
either signs of acute MI (eosinophilia, karyolysis, and leukocyte infiltration) or collagen scars compatible with an old
infarction was analyzed by examination of Van Giesonstained transverse LV sections. Infarct size was determined
planimetrically as the ratio of infarcted tissue or scar to the
length of the entire LV endocardial circumference as described previously (23). Infarcts were classified as small (4–
30%), moderate (31–49%), or large (⬎50%). Hearts that
showed no histological signs of infarction were not included
in the study (n ⫽ 7).
Assessment of apoptosis. Apoptotic cardiomyocytes were
detected with a terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay as previously
described (6, 24, 25). In brief, myocardial tissue sections were
heated in sodium citrate solution and digested with proteinase-K to expose the DNA. DNA strand breaks were labeled
with the use of terminal transferase enzyme with dUTP
molecules conjugated to alkaline phosphatase and visualized
immunohistochemically. The assay was standardized with
the use of serial sections treated with DNase I (1 U/ml for 30
min at 37°C) to induce the formation of DNA strand breaks
(positive control of apoptosis). The development of the immunohistochemical staining was monitored by microscopy, and
the reaction was interrupted at the moment when intense
positive signal appeared in the corresponding DNase
I-treated section (25).
Analysis of apoptosis was performed in one LV section
obtained from the sample that showed maximal infarct size.
The number of apoptotic cardiomyocytes was counted in the
whole LV under light microscopy with an ocular grid. The
cardiomyocyte origin of the apoptotic cells was identified by
the presence of myofilaments surrounding the nucleus. The
amounts of apoptotic cardiomyocytes were expressed as the
proportion of the TUNEL-positive cardiomyocyte nuclei from
the total number of cardiomyocyte nuclei, which was obtained by multiplying the density of cardiomyocyte nuclei in
the serial DNase I-treated control section times the area of
the section. The density of cardiomyocyte nuclei was always
counted in at least four representative microscopic fields in
each region of interest. To verify that all cardiomyocyte
nuclei were labeled in DNase I-treated sections, we compared
the nuclear densities obtained in DNase I-treated sections
and Hoechst 33258-stained serial sections (n ⫽ 10). We did
not find significant difference in the average nuclear density
obtained with these two methods [887 ⫾ 200 vs. 773 ⫾ 98
(SD)]. The proportion of apoptotic cardiomyocytes was
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APOPTOSIS AND VENTRICULAR REMODELING
Fig. 1. A: terminal deoxynucleotide transferase-mediated dUTP
nick-end labeling (TUNEL)-positive cardiomyocytes in the border
zone (BZ) of an infarcted tissue 24 h after coronary occlusion, an area
marked with myocardial infarction (MI). B: single scattered apoptotic cardiomyocytes were observed in the BZ of the infarct scars (see
also BZ in C) as well as in the remote noninfarcted myocardium. C:
at later time points, TUNEL-positive cardiomyocytes were not found
among the fibrous infarct scar (shown as MI). TUNEL-positive cardiomyocytes contained condensed nuclei, which is typical of apoptosis as shown in A and B.
central infarcted areas (average 1.44 ⫾ 0.99%). Apoptosis remained high in the myocardium bordering infarct scars at 1 wk (0.23 ⫾ 0.09, P ⫽ 0.01), 4 wk (0.34 ⫾
0.20%, P ⬍ 0.01), and even 12 wk (0.10 ⫾ 0.05%) after
infarction. The percentages of apoptotic cardiomyocytes tended to be higher in the border zone areas of
moderate- and large-sized infarctions than in the border zones of small-sized infarctions at 24 h (5.56 vs.
3.36%) and 1 wk (0.29 vs. 0.18%) after infarction. The
percentages were similar 4 and 12 wk after infarction
at 0.33 versus 0.37% and 0.10 versus 0.09%, respectively.
In the remote noninfarcted myocardium, apoptosis
significantly increased already on day 1 after coronary
occlusion (0.07 ⫾ 0.03%, P ⫽ 0.02) and again 4 wk after
infarction (0.09 ⫾ 0.05%, P ⬍ 0.01) when compared
with controls (Fig. 2). Again, the percentages of apoptosis in rats with large- and moderate-sized infarctions
tended to be higher than in those with small infarctions at 24 h (0.08 vs. 0.04%), 1 wk (0.05 vs. 0.02%), and
4 wk (0.10 vs. 0.07%). However, 12 wk later, infarction
percentages were similar (0.04%) in all sizes. There
were no predilection sites of apoptotic cells other than
the border zone area.
Relative distributions of apoptotic cells in infarcted
border zone and remote areas. To further analyze the
quantitative distribution of apoptosis in the remote,
border zone and infarcted areas, we determined the
proportion of each region from the total area of the LV
section. We then calculated the distribution of apoptotic cells between these areas. On day 1 after coronary
artery ligation, most of the TUNEL positivity occurred
in the infarcted and border zone tissues (26 and 67%
vs. 7% in the remote areas) (Fig. 3). Thereafter, the
relative share of apoptotic cells in the remote areas
gradually increased to 37% in 1 wk (P ⬍ 0.01 vs. day 1),
Fig. 3. Relative proportions of apoptotic cardiomyocytes in the remote, BZ, and MI areas. On day 1 after infarction, majority of
apoptosis occurred in the MI and BZ areas (open and solid bars,
respectively). Thereafter, the relative proportion of apoptotic cells
found in the remote noninfarcted tissue (hatched bars) significantly
increased when compared with the BZ tissue (solid bars). **P ⬍ 0.01.
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Fig. 2. Percentages of cardiomyocyte apoptosis in infarcted rats (n ⫽
7, 8, 11, and 8 at 24 h, 1 wk, 4 wk, and 12 wk after MI, respectively)
and sham-operated rats (n ⫽ 8, 14, 8, and 6 after 24 h, 1 wk, 4 wk,
and 12 wk followup, respectively). A: apoptosis peaked on day 1 after
MI in the infarcted and BZ tissues (cross hatched and solid bars,
respectively) but was also increased in the remote noninfarcted
myocardium (hatched bars) when compared with the sham-operated
animals (open bars). B: apoptosis remained high thereafter in the BZ
of the infarction scars until 4 wk after MI. Four weeks after MI,
apoptosis was also increased in the remote noninfarcted myocardium. Note logarithmic y-axis scale in A. *P ⬍ 0.05; **P ⬍ 0.01.
APOPTOSIS AND VENTRICULAR REMODELING
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Fig. 4. Echocardiographic measurements of ejection fraction (EF, A) and left ventricular (LV)
diastolic diameter (LVDD, B). Sham-operated
animals showed only minor changes in the EF or
the LVDD (dotted lines). After MI, the EF decreased already at 24 h and remained low at
later time points (solid line). LV diameter increased gradually after MI (solid line) and was
significantly higher at 1 and 4 wk. bl, baseline.
*P ⬍ 0.05 vs. baseline; **P ⬍ 0.01 vs. baseline.
DISCUSSION
Occlusion of a major coronary artery in the rat is a
well-characterized animal model of acute MI and its
chronic sequelae, including congestive heart failure
(19, 22, 23, 28). Previous studies in this model have
shown that internucleosomal DNA fragmentation and
TUNEL-positive cardiomyocytes can be found in the
central ischemic areas in the acute phase of infarction
(5, 13). Moreover, activated forms of caspases (2), the
key executioners of apoptotic cell death, have been
found in myocytes in this situation. Increased expression of the apoptosis-mediating Fas receptor (13) and
increased ratio of a proapoptotic protein Bax to an
antiapoptotic protein Bcl-2 have been suggested as
potential mediators of myocyte apoptosis in this model
(3).
On the basis of quantification of TUNEL positivity,
apoptosis has been suggested to be the major form of
cell death during the first hours of evolution of MI (5,
13). Apoptosis has also been found to a smaller extent
in the viable myocardium of the LV free wall (3). In this
area, apoptosis peaked on day 1 after infarction but
continued at a low rate (0.02 apoptotic nuclei per 100
cardiomyocytes) until 4 wk after infarction (3). In contrast, the remote myocardium of interventricular septum showed constantly low rates of apoptosis (⬍0.01
apoptotic nuclei per 100 cardiomyocytes) (3). We extend the previous findings of Cheng et al. (3) by showing that the occurrence of cardiomyocyte apoptosis in
Fig. 5. The proportion of cardiomyocyte apoptosis in the BZ areas (A) and the remote myocardium (B) compared with the relative change in
LV diameter from baseline as measured with
echocardiography 1 and 4 wk after MI. In remote
areas, the amount of apoptosis correlated with
ventricular enlargement (r ⫽ 0.60; P ⬍ 0.01).
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54% in 4 wk (P ⬍ 0.01 vs. day 1), and 70% in 12 wk (P ⬍
0.01 vs. day 1) after infarction. Thus, although the
percentage of apoptotic cells was higher in the border
zone areas, the remote areas dominated the actual
amount of apoptosis from week 4 after infarction.
Apoptosis, ventricular function, and ventricular diameter. To characterize the changes in ventricular
function and geometry after MI, we measured EF and
LVDD with the use of echocardiography preoperatively
and at 24 h, 1 wk, and 4 wk after the operation (Fig. 4).
The sham-operated animals showed only minor
changes in EF during the study (Fig. 4A). In contrast,
EF decreased already 24 h after coronary occlusion (63
vs. 51%, P ⫽ 0.01) and remained low thereafter. Shamoperated animals did not show significant increases in
the LVDD (Fig. 4B). After coronary ligation, the ventricular diameters were highly variable (from 7.5 to
11.1 mm). The average ventricular diameter gradually
increased and was 12% higher compared with baseline
at 4 wk after infarction (P ⫽ 0.01).
We did not find any correlation between the EF and
the amount of apoptosis either in the border zones or
remote myocardium after infarction. Neither was there
a correlation between the amount of apoptosis in the
border zone areas and the change in the ventricular
diastolic diameter. In contrast, the amount of apoptosis
in the remote myocardium was related to the increase
of LVDD (r ⫽ 0.60; P ⬍ 0.01) (Fig. 5B). The high degree
of ventricular enlargement was associated with a high
percentage of apoptosis.
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APOPTOSIS AND VENTRICULAR REMODELING
correlated with an increase in the ventricular diameter
1 and 4 wk after infarction. This provides evidence that
apoptosis in this region plays a role in post-MI ventricular remodeling and thus may contribute to the development of congestive heart failure. Actually, we calculated that by 4 wk after infarction the majority of
apoptosis occurs in this region due to the large volume
of the remote myocardium when compared with the
border zone (Fig. 3).
There are several possible triggers of apoptosis after
MI in the remote myocardium. It has been proposed
that stretch and tension of ventricular wall due to
increased filling pressure could have caused apoptosis
in rats with large infarctions (3). Notably, ␤-adrenoceptor blocking agents have reduced cardiomyocyte
apoptosis in such experiments (31). In the present
study, the average size of infarction was moderate and
did not cause severe, progressive impairment of ventricular function, as measured with echocardiography.
The fact that there was no correlation between the
amount of apoptosis and the decrease in EF may be
partly due to the technical limitations of assessing the
overall function of the ventricle with the use of single
plane M-mode echocardiography.
Some paracrine mediators that are actively produced
in the remote myocardium after infarction in rats, such
as tumor necrosis factor-␣ (11) and angiotensin II (10),
have been shown to induce cardiomyocyte apoptosis in
vitro (14, 16). Recently, angiotensin-converting enzyme
inhibitors have been shown to decrease apoptosis in
the border zones of infarct scars in a dog model of
ischemic heart failure (9). However, the contributions
of afterload reduction and direct action on cardiac
tissue angiotensin production to the attenuation of
remodeling by angiotensin-converting inhibitors remain obscure (29).
In conclusion, we have shown that cardiomyocyte
apoptosis occurs after MI in the rat continuously over
an extended period of time both in the viable border
zones of infarct scars and in the remote noninfarcted
myocardium. In the remote myocardium, apoptosis correlates with ventricular enlargement and thus plays a
role in the postinfarction remodeling in this model.
We thank Terhi Ilomäki for technical assistance and HewlettPackard (Espoo, Finland) for providing the ultrasound machine.
This study was supported by the Aarne Koskelo Foundation, Ida
Montin Foundation, Finnish Heart Association, Finnish Cultural
Foundation, Sigrid Jusélius Foundation, and by the clinical research
funds of the Helsinki and Turku University Central Hospitals.
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